Molecular Biology

By David P. Clark and Nanette J. Pazdernik

Molecular Biology, Second Edition, examines the basic concepts of molecular biology while incorporating primary literature from today’s leading researchers. This updated edition includes Focuses on Relevant Research sections that integrate primary literature from Cell Press and focus on helping the student learn how to read and understand research to prepare them for the scientific world.The new Academic Cell Study Guide features all the articles from the text with concurrent case studies to help students build foundations in the content while allowing them to make the appropriate connections to the text. Animations provided deal with topics such as protein purification, transcription, splicing reactions, cell division and DNA replication and SDS-PAGE. The text also includes updated chapters on Genomics and Systems Biology, Proteomics, Bacterial Genetics and Molecular Evolution and RNA. An updated ancillary package includes flashcards, online self quizzing, references with links to outside content and PowerPoint slides with images.This text is designed for undergraduate students taking a course in Molecular Biology and upper-level students studying Cell Biology, Microbiology, Genetics, Biology, Pharmacology, Biotechnology, Biochemistry, and Agriculture.

• NEW: "Focus On Relevant Research" sections integrate primary literature from Cell Press and focus on helping the student learn how to read and understand research to prepare them for the scientific world
• NEW: Academic Cell Study Guide features all articles from the text with concurrent case studies to help students build foundations in the content while allowing them to make the appropriate connections to the text
• NEW: Animations provided include topics in protein purification, transcription, splicing reactions, cell division and DNA replication and SDS-PAGE
• Updated chapters on Genomics and Systems Biology, Proteomics, Bacterial Genetics and Molecular Evolution and RNA
• Updated ancillary package includes flashcards, online self quizzing, references with links to outside content and PowerPoint slides with images
• Fully revised art program

• Language: English
• Publisher: Elsevier Science
• Release date: Mar 20, 2012
• ISBN: 9780123785954

David P. Clark

David P. Clark did his graduate work on bacterial antibiotic resistance to earn his Ph.D. from Bristol University, England. He later crossed the Atlantic to work as a postdoctoral researcher at Yale University and then the University of Illinois. Dr Clark recently retired from teaching Molecular Biology and Bacterial Physiology at Southern Illinois University which he joined in 1981. His research into the Regulation of Alcohol Fermentation in E. coli was funded by the U.S. Department of Energy, from 1982 till 2007. In 1991 he received a Royal Society Guest Research Fellowship to work at Sheffield University, England while on sabbatical leave.

Book preview

UNIT 1

Basic Chemical and Biological Principles

Chapter 1 Cells and Organisms

Chapter 2 Basic Genetics

Chapter 3 DNA, RNA, and Protein

Chapter 4 Genomes and DNA

Chapter 5 Manipulation of Nucleic Acids

Chapter 1

Cells and Organisms

1. What Is Life? 

2. Living Creatures Are Made of Cells 

3. Eubacteria and Archaea Are Genetically Distinct 

4. Eukaryotic Cells Are Subdivided into Compartments 

5. The Diversity of Eukaryotes 

6. Haploidy, Diploidy, and the Eukaryote Cell Cycle 

7. Organisms Are Classified 

8. Some Widely-Studied Organisms Serve as Models 

9. Basic Characteristics of a Model Organism 

10. Purifying DNA from Model Organisms 

11. Viruses Are Not Living Cells 

12. Bacterial Viruses Infect Bacteria 

13. Human Viral Diseases Are Common 

14. A Variety of Subcellular Genetic Entities Exist 

Before tackling the complex details of biology at the molecular level, we need to get familiar with the subject of our investigation—the living world. First, we will consider what it means to be alive and then we shall survey a range of cells and organisms that are often studied by molecular biologists.

Life is impossible to define exactly, but a general idea is sufficient here. Living things consist of cells—some consist of a single cell, whereas others are made from assemblies of many million cells. Whatever the situation, living cells must grow, divide, and pass on their characteristics to their offspring. Molecular biology focuses on the details of growth and division. In particular, we are interested in how division is arranged so that each descendent can inherit their parents characteristics.

Scientists have devoted much effort in investigating certain favored organisms. In some cases this is a matter of convenience—bacteria, yeast, and other single-celled microorganisms are relatively easy to investigate. In other cases it is due to self-interest. Mice—and some other animals—reveal much about humans, plants provide our food, and viruses make us sick.

1. What Is Life?

Although there is no definition of life that suits all people, everyone has an idea of what being alive means. Generally, it is accepted that something is alive if it can grow and reproduce, at least during some stage of its existence. Thus, we still regard adults who are no longer growing and those individuals beyond reproductive age as being alive. We also regard sterile individuals, such as mules or worker bees as being alive, even though they lack the ability to reproduce. Part of the difficulty in defining life is the complication introduced by multicellular organisms. Although a multicellular organism as a whole may not grow or reproduce some of its cells may still retain these abilities.

No satisfactory technical definition of life exists. Despite this we understand what life entails. In particular, life involves a dynamic balance between duplication and alteration.

The basic ingredients needed to sustain life include the following:

• Genetic information Biological information is carried by the nucleic acid molecules, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The units of genetic information are known as genes, and each consists physically of a segment of a nucleic acid molecule. DNA is used for long-term storage of large amounts of genetic information (except by some viruses—see Ch. 21). Whenever genetic information is actually used, working copies of the genes are carried on RNA. The total genetic information possessed by an organism is known as its genome. DNA genomes are maintained by replication, a process where a copy of the DNA is produced by enzymes and then passed on to the daughter cells.

• Mechanism for energy generation By itself, information is useless. Energy is needed to put the genetic information to use. Living creatures must all obtain energy for growth and reproduction. Metabolism is the set of processes in which energy is acquired, liberated, and used for biosynthesis of cell components, then catabolized and recycled. Living organisms use raw material from the environment to grow and reproduce.

• Machinery for making more living matter Synthesis of new cell components requires chemical machinery. In particular, the ribosomes are needed for making proteins, the macromolecules that make up the bulk of all living tissue. These tiny subcellular machines allow the organism to grow and maintain itself.

• A characteristic outward physical form Living creatures all have a material body that is characteristic for each type of life-form. This structure contains all the metabolic and biosynthetic machinery for generating energy and making new living matter. It also contains the DNA molecules that carry the genome. The form has levels of organization from single cells to tissues, organs, and organ systems of multicellular life-forms.

• Identity or Self All living organisms have what one might call an identity. The term self-replication implies that an organism knows to make a copy of itself—not merely to assemble random organic material. This concept of self versus non-self is very evident in the immune systems that protect higher animals against disease. But even primitive creatures attempt to preserve their own existence.

• Ability to reproduce The organism uses energy and raw materials to make itself, and then uses the same materials to produce offspring. Some organisms simply reproduce with asexual reproduction (making offspring without creating gametes) and other organisms use sexual reproduction (two gametes fuse to form a new organism).

• Adaptation The most important characteristic of a living creature is the ability to adapt to its current environment. This concept also encompasses evolution or the adaptations that get passed from generation to generation.

2. Living Creatures Are Made of Cells

Looking around at the living creatures that inhabit this planet, one is first struck by their immense variety: squids, seagulls, sequoias, sharks, sloths, snakes, snails, spiders, strawberries, soybeans, Saccharomyces cerevisiae, and so forth. Although highly diverse to the eye, the biodiversity represented by these creatures is actually somewhat superficial. The most fascinating thing about life is not its superficial diversity but its fundamental unity. All of these creatures, together with microscopic organisms too small to see with the naked eye, are made up of cells, structural units or compartments that have more or less the same components.

Matter is divided into atoms. Genetic information is divided into genes. Living organisms are divided into cells.

The idea that living cells are the structural units of life was first proposed by Schleiden and Schwann in the 1830s. Cells are microscopic structures that vary considerably in shape. Many are spherical, cylindrical, or roughly cuboidal, but many other shapes are found, such as the long-branched filaments of nerve cells. Many microscopic life-forms consist of a single cell, whereas creatures large enough to see usually contain thousands of millions. Each cell is enclosed by a cell membrane composed of proteins and phospholipids and contains a complete copy of the genome (at least at the start of its life). Living cells possess the machinery to carry out metabolic reactions and generate energy and are usually able to grow and divide. Moreover, living cells always result from the division of pre-existing cells, and they are never assembled from their component parts. This implies that living organisms too can only arise from pre-existing organisms. In the 1860s, Louis Pasteur confirmed experimentally that life cannot arise spontaneously from organic matter. Sterilized nutrient broth did not spoil or go bad unless it was exposed to microorganisms in the air.

In most multicellular organisms, the cells are specialized in a variety of ways (Fig. 1.01). The development of specialized roles by particular cells or whole tissues is referred to as differentiation. For example, the red blood cells of mammals lose their nucleus and the enclosed DNA during development. Once these cells are fully differentiated, they can perform only their specialized role as oxygen carriers and can no longer grow and divide. Some specialized cells remain functional for the life span of the individual organism, whereas others have limited life spans, sometimes lasting only a few days or hours. For multicellular organisms to grow and reproduce, some cells clearly need to keep a complete copy of the genome and retain the ability to become another organism. The other differentiated cells perform their function, but do not retain the ability to create an entirely new organism. In single-celled organisms, such as bacteria or protozoa, each individual cell has a complete genome and can grow and reproduce; hence, every cell is essentially the same.

Figure 1.01 Some Cells Differentiate

Cells differentiate into all different shapes and sizes to provide specialized functions in a multicellular organism. In this figure, red blood cells (A) are specialized to exchange carbon dioxide and oxygen in the tissues of humans; fibroblasts (B) provide support to various organs; and neurons (C) transmit signals from the environment to the brain to elicit a response.(Credit: A) Esposito, et al. (2010) Biophysical J 99(3): 953–960. B) Dokukina and Gracheva (2010) Biophysical J 98(12): 2794–2803. C) Fino and Yuste (2011) Neuron 69(6): 1188–1203.)

2.1 Essential Properties of a Living Cell

At least in the case of unicellular organisms, each cell must possess the characteristics of life as discussed above. Each living cell must generate its own energy and synthesize its own macromolecules. Each must have a genome, a set of genes carried on molecules of DNA. (Partial exceptions occur in the case of multicellular organisms, where responsibilities may be distributed among specialized cells and some cells may lack a complete genome.)

Membranes do not merely separate living tissue from the non-living exterior. They are also the site of many biosynthetic and energy-yielding reactions.

A cell must also have a surrounding membrane that separates the cell interior, the cytoplasm, from the outside world. The cell membrane, or cytoplasmic membrane, is made from a double layer of phospholipids together with proteins (Fig. 1.02). Phospholipid molecules consist of a water-soluble head group, including phosphate, found at the surface of the membrane, and a lipid portion consisting of two hydrophobic chains that form the body of the membrane (Fig. 1.03). The phospholipids form a hydrophobic layer that greatly retards the entry and exit of water-soluble molecules. For the cell to grow, it must take up nutrients. For this, transport proteins that transverse the membranes are necessary. Many of the metabolic reactions involved in the breakdown of nutrients to release energy are catalyzed by soluble enzymes located in the cytoplasm. Other energy-yielding series of reactions, such as the respiratory chain or the photosynthetic system, are located in membranes. The proteins may be within or attached to the membrane surfaces.

Figure 1.02 A Biological Membrane

A biological membrane is formed by phospholipid and protein. The phospholipid layers are oriented with their hydrophobic tails inward and their hydrophilic heads outward. Proteins may be within the membrane (integral) or lying on the membrane surfaces.

Figure 1.03 Phospholipid Molecules

Phospholipd molecules of the kind found in membranes have a hydrophilic head group attached via a phosphate group to glycerol. Two fatty acids are also attached to the glycerol via ester linkages.

The cytoplasmic membrane is physically weak and flexible. Many cells therefore have a tough structural layer, the cell wall, outside the cell membrane. Most bacterial and plant cells have hard cell walls, though animal cells usually do not. Thus, a cell wall is not an essential part of a living cell.

Based on differences in compartmentalization, living cells may be divided into two types, the simpler prokaryotic cell and the more complex eukaryotic cell. By definition, prokaryotes are those organisms whose cells are not subdivided by membranes into a separate nucleus and cytoplasm. All prokaryote cell components are located together in the same compartment. In contrast, the larger and more complicated cells of higher organisms (animals, fungi, plants, and protists) are subdivided into separate compartments and are called eukaryotic cells. Figure 1.04 compares the design of prokaryotic and eukaryotic cells.

Figure 1.04 Typical Prokaryotic Cell

The components of a typical prokaryote, a bacterium, are depicted. There is no nucleus and the DNA is free in the cytoplasm where it is compacted into the nucleoid.

Another property of living cells are soluble enzymes located in the cytoplasm. Cellular enzymes catalyze biosynthesis of the low molecular weight precursors to protein and nucleic acids. Protein synthesis requires a special organelle, the ribosome. This is a subcellular machine that consists of several molecules of RNA and around 50 proteins. It uses information that is carried from the genome by special RNA molecules, known as messenger RNA. The ribosome decodes the nucleic acid-encoded genetic information on the messenger RNA to make protein molecules.

Some single-celled protozoa, such as Paramecium, have multiple nuclei within each single cell. In addition, in certain tissues of some multicellular organisms several nuclei may share the same cytoplasm and be surrounded by only a single cytoplasmic membrane. Such an arrangement is known as a syncytium when it is derived from multiple fused cells.

2.2 Prokaryotic Cells Lack a Nucleus

Bacteria (singular, bacterium) are the simplest living cells and are classified as prokaryotes. Bacterial cells (Fig. 1.05) are always surrounded by a membrane (the cell or cytoplasmic membrane) and usually also by a cell wall. Like all cells, they contain all the essential chemical and structural components necessary for the type of life they lead. Typically, each bacterial cell has a single chromosome carrying a full set of genes providing it with the genetic information necessary to operate as a living organism. Most bacteria have 3,000–4,000 genes, although some have as few as 500.

Figure 1.05 Typical Eukaryotic Cell

A typical eukaryotic cell showing a separate compartment called the nucleus that contains the DNA.

The minimum number of genes needed to allow the survival of a living cell is uncertain. Mycoplasma genitalium has the smallest genome of any cultured bacterium. Its 485 genes have been systematically disrupted and around 100 are dispensable. Although this suggests around 385 genes are needed, some are duplicated in function. In addition, M. genitalium relies on a host organism to survive, and therefore, it lacks genes for making certain essential components. The entire genome of M. genitalium has recently been successfully synthesized and re-introduced into a cell in order to continue this analysis.

A typical bacterial cell, such as Escherichia coli, is rod shaped and about two or three microns long and one micron wide. Bacteria are not limited to a rod shape (Fig. 1.06); spherical, filamentous, or spirally-twisted bacteria are also found. Occasional giant bacteria occur, such as Epulopiscium fishelsoni, which inhabits the surgeonfish and measures a colossal 50 microns by 500 microns—an organism visible to the naked eye. In contrast, typical eukaryotic cells are 10 to 100 microns in diameter.

Figure 1.06 Bacillus subtilis Cells Visualized by Scanning Electron Microscopy

Scanning electron microscopy of B. subtilis shows tube shaped connections between bacterial cells.(Credit: Dubey, et al. (2011) Cell 144(4): 590–600.)

A smaller cell has a larger surface-to-volume ratio. Smaller cells transport nutrients relatively faster, per unit mass of cytoplasm (i.e., cell contents), and so can grow more rapidly than larger cells. Because bacteria are less structurally complex than animals and plants, they are often referred to as lower organisms. However, it is important to remember that present-day bacteria are at least as well adapted to modern conditions as animals and plants and are just as highly evolved as so-called higher organisms. In many ways, bacteria are not so much primitive as specialized for growing more efficiently in many environments than larger and more complex organisms.

A micrometer (μ m), also known as a micron, is a millionth of a meter (i.e., 10−6 meter).

Cells are separated from their environments by membranes. In the more complex cells of eukaryotes, the genome is separated from the rest of the cell by another set of membranes.

If higher organisms disappeared from the Earth, the prokaryotes would survive and evolve. They do not need us although we need them.

3. Eubacteria and Archaea Are Genetically Distinct

There are two distinct types of prokaryotes, the eubacteria and archaebacteria or archaea, which are no more genetically related to each other than either group is to the eukaryotes. Both eubacteria and archaea show the typical prokaryotic structure—in other words, they both lack a nucleus and other internal membranes. Thus, cell structure is of little use for distinguishing these two groups. The eubacteria include most well-known bacteria, including all those that cause disease. When first discovered, the archaea were regarded as strange and primitive. This was largely because most are found in extreme environments (Fig. 1.07) and/or possessed unusual metabolic pathways. Some grow at very high temperatures; others in very acidic conditions; and others in very high salt. The only major group of archaea found under normal conditions is the methane bacteria, which, however, have a very strange metabolism. They contain unique enzymes and co-factors that allow the formation of methane by a pathway found in no other group of organisms. Despite this, the transcription and translation machinery of archaea resembles that of eukaryotes, so they turned out to be neither fundamentally strange nor truly primitive when further analyzed.

At a fundamental level, three domains of life, eubacteria, archaea, and eukaryotes, have replaced the old-fashioned division of animal and vegetable.

Figure 1.07 Hot springs in Ethiopia

Hot springs are good sites to find archaea. These springs are in the Dallol area of the Danakil Depression, 120 meters below sea level. The Danakil Depression of Ethiopia is part of the East African Rift Valley. Hot water flows from underground to form these pools. The water is heated by volcanic activity and is at high pressure, causing minerals in the rock to dissolve in the water. The minerals precipitate out as the water cools at the surface, forming the deposits seen here.(Credit: Bernhard Edmaier, Science Photo Library.)

Biochemically, there are major differences between the eubacterial and archaeal cells. In all cells, the cell membrane is made of phospholipids, but the nature and linkage of the lipid portion is quite different in the eubacteria and archaea (Fig. 1.08). The cell wall of eubacteria is always made of peptidoglycan, a molecule unique to this group of organisms. Archaea often have cell walls, but these are made of a variety of materials in different species, and peptidoglycan is never present. Thus, the only real cellular structures possessed by prokaryotes, the cell membrane and cell wall, are in fact chemically different in these two groups of prokaryotes. The genetic differences will be discussed later when molecular evolution is considered (see Ch. 26).

Figure 1.08 Lipids of Archaea

In eubacteria and eukaryotes, the fatty acids of phospholipids are esterified to the glycerol. In archaea, the lipid portion consists of branched isoprenoid hydrocarbon chains joined to the glycerol by ether linkages (as shown here). Such lipids are much more resistant to extremes of pH, temperature, and ionic composition.

4. Eukaryotic Cells Are Subdivided into Compartments

A eukaryotic cell has its genome inside a separate compartment, the nucleus. In fact, eukaryotic cells have multiple internal cell compartments surrounded by membranes. The nucleus itself is surrounded by a double membrane, the nuclear envelope, which separates the nucleus from the cytoplasm, but allows some communication with the cytoplasm via nuclear pores (Fig. 1.09). The genome of eukaryotes consists of 10,000–50,000 genes carried on several chromosomes. Eukaryotic chromosomes are linear, unlike the circular chromosomes of bacteria. Most eukaryotes are diploid, with two copies of each chromosome. Consequently, they possess at least two copies of each gene. In addition, eukaryotic cells often have multiple copies of certain genes as the result of gene duplication.

Figure 1.09 A Eukaryote Has Multiple Cell Compartments

False color transmission electron micrograph of a plasma cell from bone marrow. Multiple compartments surrounded by membranes, including a nucleus, are found in eukaryotic cells. Characteristic of plasma cells is the arrangement of heterochromatin (orange) in the nucleus, where it adheres to the inner nuclear membrane. Also typical is the network of rough endoplasmic reticulum (yellow dotted lines) in the cytoplasm. The oval or rounded crimson structures in the cytoplasm are mitochondria. Magnification×4,500.(Credit: Dr. Gopal Murti, Science Photo Library.)

Eukaryotes possess a variety of other organelles. These are subcellular structures that carry out specific tasks. Some are separated from the rest of the cell by membranes (so-called membrane-bound organelles) but others (e.g., the ribosome) are not. The endoplasmic reticulum is a membrane system that is continuous with the nuclear envelope and permeates the cytoplasm. The Golgi apparatus is a stack of flattened membrane sacs and associated vesicles that is involved in secretion of proteins, or other materials, to the outside of the cell. Lysosomes are membrane-bound structures containing degradative enzymes and specialized for digestion.

All except a very few eukaryotes contain mitochondria (singular, mitochondrion; Fig. 1.10). These are generally rod-shaped organelles, bounded by a double membrane. They resemble bacteria in their overall size and shape. As will be discussed in more detail (see Ch. 4), it is thought that mitochondria are indeed evolved from bacteria that took up residence in the primeval ancestor of eukaryotic cells. Like bacteria, mitochondria each contain a circular molecule of DNA. The mitochondrial genome is similar to a bacterial chromosome, though much smaller. The mitochondrial DNA has some genes needed for mitochondrial function.

Figure 1.10 Mitochondrion

A mitochondrion is surrounded by two concentric membranes. The inner membrane is folded inward to form cristae. These are the site of the respiratory chain that generates energy for the cell.

Mitochondria are specialized for generating energy by respiration and are found in all eukaryotes. (A few eukaryotes are known that cannot respire; nonetheless, these retain remnant mitochondrial organelles—see below.) In eukaryotes, the enzymes of respiration are located on the inner mitochondrial membrane, which has numerous infoldings to create more membrane area. This contrasts with bacteria, where the respiratory chain is located in the cytoplasmic membrane, as no mitochondria are present.

Chloroplasts are membrane-bound organelles specialized for photosynthesis (Fig. 1.11). They are found only in plants and some single-celled eukaryotes. They are oval- to rod-shaped and contain complex stacks of internal membranes that contain the green, light-absorbing pigment chlorophyll and other components needed for trapping light energy. Like mitochondria, chloroplasts contain a circular DNA molecule and are thought to have evolved from a photosynthetic bacterium.

Figure 1.11 Chloroplast

The chloroplast is bound by a double membrane and contains infolded stacks of membrane specialized for photosynthesis. The chloroplast also contains ribosomes and DNA.

Eukaryotic cells have extensive intracellular architecture to maintain their shape and move materials and organelles around the cells. The cytoskeleton is a complex network of filaments made of proteins like actin, vinculin, and fibronectin (Fig. 1.12). Besides maintaining cell shape, the cytoskeleton is important for cellular transport. For example, cytoskeletal fibers run through the long axons of neurons, and vesicles filled with neurotransmitters travel up and down the axon to facilitate the communication between the nucleus and the nerve fibers. The cytoskeleton also initiates cellular movements. By increasing the length of fibers on one side of the cell and decreasing their length on the opposite side, the cell can physically move. This is especially true for smaller single-cell eukaryotes and for movements during a multicellular organism’s development. Finally, these cytoskeletal movements are important to processes like cell division, since the very same fibers make up the spindle.

Life is modular. Complex organisms are subdivided into organs. Large and complex cells are divided into organelles.

Eukaryotes have many membrane-bound organelles to perform functions like respiration (mitochondria), enzyme degradation (lysosomes), and protein processing and secretion (Golgi apparatus and endoplasmic reticulum).

Eukaryotic cells have internal structural elements called a cytoskeleton.

Figure 1.12 Cytoskeleton

Actin, vinculin, and fibronectin are three cytoskeletal proteins that give this cell a flattened edge. This edge has adhesions that connect the cell to the dish in vitro, but function to keep the cell attached to other cells within the organs of a multicellular organism.(Credit: Byron, et al. (2010) Curr Biol 20(24): R1063–R1067)

5. The Diversity of Eukaryotes

Unlike prokaryotes that fall into two distinct genetic lineages (the eubacteria and archaebacteria), all eukaryotes are genetically related, in the sense of being ultimately derived from the same ancestor. Perhaps this is not surprising since all eukaryotes share many advanced features that the prokaryotes lack. When it is said that all eukaryotes are genetically related, this refers to the nuclear part of the eukaryotic genome, not the mitochondrial or chloroplast DNA molecules that have become part of the modern eukaryotic cell.

Box 1.01 Anaerobic Eukaryote

Some single-celled eukaryotes lack true respiratory mitochondria and must grow by fermentation. For example, Entamoeba histolytica invades and destroys the tissues of the intestines, causing amoebic dysentery (Fig. 1.13). It may spread to the liver causing abscesses to develop. This infection is acquired from flies, or by contaminated food or water.

Figure 1.13 Entamoeba: An Anaerobic Eukaryote

Endocytosis in Entamoeba histolytica—Phase contrast (a) and acridine orange fluorescence (b) images showed many acidic vesicles and vacuoles in the cytoplasm of Entamoeba. Ultrathin section (c) of a trophozoite confirmed that the cytoplasm is filled with vacuoles (v); gold-labeled lactoferrin could be found bound to parasite surface (arrowheads in d), inside peripheral tubules (arrowheads in e) and vesicles (arrowheads in e). Bars: 17 μm (a, b); 1.6 μm (c); 400 nm (d); 500 nm (e); 170 nm (f).(Credit: de Souza, et al. (2009) Progress in Histochem Cytochem 44(2): 67–124.)

A wide variety of eukaryotes live as microscopic single cells. However, the most visible eukaryotes are larger multicellular organisms that are visible to the naked eye. Traditionally, these higher organisms have been divided into the plant, fungus, and animal kingdoms. This classification must be modified to include several new groups to account for the single-celled eukaryotes. Some single-celled eukaryotes may be viewed as plants, fungi, or animals. Others are intermediate or possess a mixture of properties and need their own miniature kingdoms.

6. Haploidy, Diploidy, and the Eukaryote Cell Cycle

Most bacteria are haploid, having only one copy of each gene. Eukaryotes are normally diploid, having two copies of each gene carried on pairs of homologous chromosomes. While this is true of the majority of multicellular animals and many single-celled eukaryotes, there are significant exceptions. Many plants are polyploid, especially angiosperms (flowering plants). About half of the present-day angiosperms are thought to be polyploid, especially tetraploid or hexaploid. For example, coffee (ancestral haploid number=11) exists as variants with 22, 44, 66, or 88 chromosomes (i.e., 2n, 4n, 6n, and 8n). Polyploid plants have larger cells, and the plants themselves are often larger. In particular, polyploids have often been selected among domesticated crop plants, since they tend to give bigger plants with higher yields (Table 1.01).

Table 1.01. Polyploidy in Crop Plants

Polyploidy is unusual in animals, being found in occasional insects and reptiles. So far the only polyploid mammal known is a rat from Argentina that was discovered to be tetraploid in 1999. It actually has only 102 chromosomes, having lost several from the original tetraploid set of 4n=112. The tetraploid rat has larger cells than its diploid relatives. The only haploid animal known is an arthropod, a mite, Brevipalpus phoenicis, which was discovered in 2001. Infection of these mites by an endosymbiotic bacterium causes feminization of the males. The genetic females of this species reproduce by parthenogenesis (i.e., development of unfertilized eggs into new individuals).

In most animals, only the gametes, the egg and sperm cells, are haploid. After mating, two haploid gametes fuse to give a diploid zygote that develops into a new animal. However, in plants and fungi, haploid cells often grow and divide for several generations before producing the actual gametes. It seems likely that in the ancestral eukaryote a phase consisting of haploid cells alternated with a diploid phase. In yeasts, both haploid and diploid cells may be found and both types grow and divide in essentially the same manner (see above). In lower plants, such as mosses and liverworts, the haploid phase, or gametophyte, may even form a distinct multicellular plant body.

During animal development, there is an early division into germline and somatic cells. Only cells from the germline can form gametes and contribute to the next generation of animals. Somatic cells have no long-term future but grow and divide only as long as the individual animal continues to live. Hence, genetic defects arising in somatic cells cannot be passed on through the gametes to the next generation of animals. However, they may be passed on to other somatic cells. Such somatic inheritance is of great importance as it provides the mechanism for cancer. In plants and fungi there is no rigid division into germline and somatic cells. The cells of many higher plants are totipotent. In other words, a single cell from any part of the plant has the potential to develop into a complete new plant, which can develop reproductive tissues and produce gametes. This is not normally possible for animal cells. (The experimental cloning of animals such as Dolly the sheep is an artificial exception to this rule.)

Many eukaryotes alternate between haploid and diploid phases. However, the properties and relative importance of the two phases varies greatly with the organism.

The concept of germline versus somatic cells applies to animals but not to other higher organisms.

7. Organisms Are Classified

Living organisms are referred to by two names, both printed in italics; for example, Escherichia coli or Saccharomyces cerevisiae. The first name refers to the genus (plural, genera), a group of closely-related species. After its first use in a publication, the genus name is often abbreviated to a single letter, as in "E. coli." After the genus, the scientific name contains the species, or individual, name. The genus and species are the smallest subdivision of the system of biological classification. Classification of living organisms facilitates the understanding of their origins and the relationships of their structure and function. In order to classify organisms, they are first assigned to one of the three domains of life, which are eubacteria, archaea, and eukaryotes. Next, the domains are divided into kingdoms. Within domain Eukarya, there are four kingdoms:

• Protista—An artificial accumulation of primitive, mostly single-celled eukaryotes often referred to as protists that don’t belong to the other three main kingdoms. There are several groups that are distinct enough that some scientists would elevate them in rank to miniature kingdoms.

• Plants—Possess both mitochondria and chloroplasts and are photosynthetic. Typically, they are non-mobile and have rigid cell walls made of cellulose.

• Fungi—Possess mitochondria but lack chloroplasts. Once thought to be plants that had lost their chloroplasts, it is now thought they never had them. Their nourishment comes from decaying biomatter. Like plants, fungi are non-mobile, but they lack cellulose and their cell walls are made of chitin. They are genetically more closely related to animals than plants.

• Animals—Lack chloroplasts but possess mitochondria. Differ from fungi and plants in lacking a rigid cell wall. Many animals are mobile.

After an organism is classified into a kingdom, then each kingdom is divided into phyla (singular, phylum). For example, the animal kingdom is divided into 20–30 different phyla, including Platyhelminthes (flatworms), Nematoda (roundworms), Arthropoda (insects), and Mollusca (snails, squids, etc.). These divisions are then narrowed even further:

Phyla are divided into classes, such as mammals.

 Classes are divided into orders, such as primates.

  Orders are divided into families, such as hominids.

   Families are divided into genera, such as Homo.

    Genera are divided into species, such as Homo sapiens

Biological classification attempts to impose a convenient filing system upon organisms related by continuous evolutionary branching. The current tree of life can be investigated at http://www.tolweb.org.

Box 1.02 Classification Summary

Domain

 Kingdom

  Phylum

   Class

    Order

     Family

Genus

species

8. Some Widely-Studied Organisms Serve as Models

With so much biological diversity, biologists concentrate their attention on certain living organisms, either because they are convenient to study or are of practical importance, and they are termed model organisms. Although scientists like to think that the model organism is representative of all bacteria, humans, or plants, model organisms are atypical in some respects. For example, few bacteria grow as fast as E. coli and few mammals breed as fast as mice. Nonetheless, information discovered in such model systems is assumed to apply also to related organisms. In practice this often proves to be true, at least to a first approximation. Model organisms are useful to the discovery of basic principles of biology. They help researchers gain knowledge that advances medicine and agriculture. But since they do have limitations; ultimately, human cells and agriculturally-useful animals and plants have to be studied directly.

8.1 Bacteria Are Used for Fundamental Studies of Cell Function

Most of the early experiments providing the basis for modern-day molecular biology were performed using bacteria such as E. coli (see below), because they are relatively simple to analyze. Some advantages of using bacteria to study cell function are:

1. Bacteria are single-celled microorganisms. Furthermore, a bacterial culture consists of many identical cells due to lack of sexual recombination during cell division. In contrast, in multicellular organisms, even an individual tissue or organ contains many different cell types. All the cells in a bacterial culture respond in a reasonably similar way, whereas those from a higher organism will give a variety of responses, making analysis much more difficult.

2. The most commonly used bacteria have about 4,000 genes as opposed to higher organisms, which have up to 50,000. Furthermore, different selections of genes are expressed in the different cell types of a single multicellular organism.

3. Bacteria are haploid, having only a single copy of most genes, whereas higher organisms are diploid, possessing at least two copies of each gene. Analyzing genes present in a single copy is far easier than trying to analyze two different alleles of the same gene simultaneously.

4. In addition to their main chromosome, some bacteria harbor small rings of DNA called plasmids, which naturally carry extra genes. Plasmids have been used extensively by scientists as vectors to carry different genes from different organisms. Analysis of recombinant DNA in bacteria is one of the essential techniques used by molecular biologists to ascertain the function of different proteins without using the organism from which they originated.

5. Bacteria can be grown under strictly controlled conditions and many will grow in a chemically-defined culture medium containing mineral salts and a simple organic nutrient such as glucose.

6. Bacteria grow fast and may divide in as little as 20 minutes, whereas higher organisms often take days or years for each generation (Fig. 1.14).

7. A bacterial culture contains around 10⁹ cells per ml. Consequently, genetic experiments that need to analyze large numbers of cells can be done conveniently.

8. Bacteria can be conveniently stored for short periods (a couple of weeks) by placing them in the refrigerator and for longer periods (20 years or more) in low temperature freezers at −70°C. Upon thawing, the bacteria resume growth. Thus, it is not necessary to keep hundreds of cultures of bacterial mutants constantly growing just to keep them alive.

Figure 1.14 Graph of Exponential Growth of Bacterial Culture

The number of bacteria in this culture is doubling approximately every 45 minutes. This is typical for fast growing bacteria such as E. coli, which are widely used in laboratory research. The bacterial population may reach 5×10⁹ cells per ml or more in only a few hours under ideal conditions.

In practice, bacteria are usually cultured by growing them as a suspension in liquid inside tubes, flasks, or bottles. They can also be grown as colonies (visible clusters of cells) on the surface of an agar layer in flat dishes known as Petri dishes. Agar is a carbohydrate polymer extracted from seaweed that sets, or solidifies, like gelatin.

It should be noted that the convenient properties noted above apply to commonly-grown laboratory bacteria. In contrast, many bacterial species found in the wild are difficult or, by present techniques impossible, to culture in the laboratory. Many others have specialized growth requirements and most rarely grow to the density observed with the bacteria favored by laboratory researchers.

Biologists have always been pulled in two directions. Studying simple creatures allows basic principles to be investigated more easily. And yet we also want to know about ourselves.

The biological diversity of bacteria is immense since the total number of bacteria on our planet is estimated at an unbelievable 5×10³⁰. Over 90% are in the soil and subsurface layers below the oceans. The total amount of bacterial carbon is 5×10¹⁷ grams, nearly equal to the total amount of carbon found in plants. Probably over half of the living matter on Earth is microbial, yet we are still unable to identify the majority of these species. The bacteria that live in extreme environments, such as boiling hot water in the deep sea thermal vents, salty seas like the Dead Sea, and Antarctic lakes that thaw for only a few months each year, have very unique adaptations to the basic proteins that drive DNA replication, gene transcription, and protein translation. The research on these bacteria will provide a wealth of information of how life formed, exists, and persists. For example, Taq DNA polymerase, which was isolated from Thermus aquaticus, a bacterium that lives in hot springs, has extreme heat stability. This characteristic of the enzyme was exploited by scientists to artificially replicate DNA by the polymerase chain reaction, which exposes the DNA to 94°C in order to denature the helix into single strands (see Ch. 6). Basically, the genetic diversity of bacteria has barely been studied.

Box 1.03 Bacteria Have Alter Egos

E. coli is normally harmless, although occasional rogue strains occur. Even these few pathogenic E. coli strains mostly just cause diarrhea by secreting a mild form of a toxin related to that found in cholera and dysentery bacteria. However, the notorious E. coli O157:H7 carries two extra toxins and causes bloody diarrhea that may be fatal, especially in children or the elderly. In outbreaks of E. coli O157:H7, the bacteria typically contaminate ground meat used in making hamburgers. Several massive recalls of frozen meat harboring E. coli O157:H7 occurred in the late 1990s. For example, in 1997 the Hudson Foods plant in Columbus, Nebraska was forced to shut down and 25 million pounds of ground beef were recalled.

Box 1.04 Antibiotics Kill Bacteria

Patients are usually given antibiotics to treat bacterial infections. These are chemical substances capable of killing most bacteria by inhibiting specific biochemical processes, but which are relatively harmless to people. The most commonly used antibiotics, the penicillins and cephalosporins, are synthesized by a kind of fungus known as mold (Fig. 1.15). However, many antibiotics are made by one kind of bacteria in order to kill other types of bacteria. The Streptomyces group of soil bacteria produces a wide range of antibiotics including streptomycin, kanamycin, and neomycin. Some antibiotics, like chloramphenicol, were originally made by molds, but nowadays can be chemically synthesized. Finally, some antibiotics, such as sulfonamides, are entirely artificial and are only synthesized by chemical corporations.

Figure 1.15 Bacterial Growth Is Suppressed by Bread Mold

The blue mold that often grows on bread makes penicillin. When penicillin is produced by molds grown on agar in a Petri dish, it will diffuse outwards and suppress the growth of bacteria in a circle around it.

8.2 E. coli Is a Model Bacterium

Although many different types of bacteria are used in laboratory investigations, the bacterium used most often in molecular biology research is E. coli, a rod-shaped bacterium of approximately 1 by 2.5 microns. Its natural habitat is the colon (hence coli), the lower part of the large intestine of mammals, including humans. The knowledge derived by examining E. coli has been used to untangle the genetic operation of other organisms. In addition, bacteria, together with their viruses and plasmids, have been used experimentally during the genetic analysis of higher organisms.

E. coli is a gram-negative bacterium, which means that it possesses two membranes. Outside the cytoplasmic membrane possessed by all cells are the cell wall and a second, outer membrane (Fig. 1.16). (Although gram-negative bacteria do have two compartments, they are nonetheless genuine prokaryotes, as their chromosome is in the same compartment as the ribosomes and other metabolic machinery. They do not have a nucleus, the key characteristic of a eukaryote.) The presence of an outer membrane provides an extra layer of protection to the bacteria. However, it can be inconvenient to the biotechnologist who wishes to manufacture genetically-engineered proteins from genes cloned into E. coli. The outer membrane hinders protein secretion. Consequently, there has been a recent upsurge of interest in gram-positive bacteria, such as Bacillus, which lack the outer membrane.

Figure 1.16 Gram-Negative and Gram-Positive Bacteria

Gram-negative bacteria have an extra membrane surrounding the cell wall.

Box 1.05 Bacteria Can Have Sex

The famous K-12 laboratory strain of E. coli was chosen as a research tool because of its fertility. In 1946, Joshua Lederberg was attempting to carry out genetic crosses with bacteria. Until then, no mechanisms for gene transfer had been demonstrated in bacteria, and genetic crosses were therefore thought to be restricted to higher organisms. Lederberg was lucky, as most bacterial strains, including most strains of E. coli, do not mate. But among those he tested was one strain (K-12) of E. coli that happened to give positive results. Mating in E. coli K-12 is actually due to a plasmid, an extra circular molecule of DNA within the bacterium that is separate from the chromosome. Because the plasmid carries the genes for fertility, it was named the F-plasmid (see Ch. 25).

The E. coli chromosome was mapped before the advent of DNA sequencing by using conjugation experiments (see Ch. 25). The chromosome is divided into map units where the genes for thrABC are arbitrarily assigned the zero position (Fig. 1.17).

Figure 1.17 The E. coli Chromosome

The circular E. coli chromosome has been divided into 100 map units. Starting with zero at thrABC, the units are numbered clockwise from 0 to 100. Various genes are indicated with numbers corresponding to their position on the map. The replication origin (oriC) and termini (ter) of replication are also indicated. Note that chromosome replication does not start at zero map units—the zero point is an arbitrary designation.

According to Jacques Monod who discovered the operon (see Ch. 25): "What applies to E. coli applies to E. lephant."

8.3 Yeast Is a Widely-Studied Single-Celled Eukaryote

Yeast is widely used in molecular biology for many of the same reasons as bacteria. It is the eukaryote about which the most is known and the first whose genome was sequenced—in 1996. Yeasts are members of the fungus kingdom and are slightly more related to animals than plants. A variety of yeasts are found in nature, but the one normally used in the laboratory is brewer’s yeast, Saccharomyces cerevisiae (Fig. 1.18). This is a single-celled eukaryote that is easy to grow in culture. Even before the age of molecular biology, yeast was widely used as a source of material for biochemical analysis. The first enzymatic reactions were characterized in extracts of yeast, and the word enzyme is derived from the Greek for in yeast.

Figure 1.18 Yeast Cells

Colored scanning electron micrograph (SEM) of budding yeast cells (Saccharomyces cerevisiae). The smaller daughter cells are budding from the larger mother cells. Magnification:×4,000.(Credit: Andrew Syred, Science Photo Library.)

Although it is a higher organism, yeast measures up quite well to the list of useful properties that make bacteria easy to study. In addition, it is less complex genetically than many other eukaryotes. Some of its most useful attributes include:

1. Yeast is a single-celled microorganism. Like bacteria, a yeast culture consists of many identical cells. Although larger than bacteria, yeast cells are only about a tenth the size of the cells of higher animals.

2. Yeast has a haploid genome of about 12 Mb of DNA with about 6,000 genes, as compared to E. coli, which has 4,000 genes, and humans, which have approximately 25,000.

3. The natural life cycle of yeast alternates between a diploid phase and a haploid phase. Thus, it is possible to grow haploid cultures of yeast, which, like bacteria, have only a single copy of each gene, making research interpretations easy.

4. Unlike many higher organisms, yeast has relatively few of its genes—about 5%—interrupted by intervening sequences, or introns.

5. Yeast can be grown under controlled conditions in a chemically-defined culture medium and forms colonies on agar like bacteria.

6. Yeast grows fast, though not as fast as bacteria. The cell cycle takes approximately 90 minutes (compared to around 20 minutes for fast-growing bacteria).

7. Yeast cultures can contain around 10⁹ cells per ml of culture media, like bacteria.

8. Yeast can be readily stored at low temperatures.

9. Genetic analysis using recombination is much more powerful in yeast than in higher eukaryotes. Furthermore, collections of yeast strains that each have one yeast gene deleted are available.

Yeast may grow as diploid or haploid cells (Fig. 1.19). Both haploid and diploid yeast cells grow by budding, rather than symmetrical cell division. In budding, a bulge, referred to as a bud, forms on the side of the mother cell. The bud gets larger and one of the nuclei resulting from nuclear division moves into the bud. Finally, the cross wall develops and the new cell buds off from the mother. Especially under conditions of nutritional deprivation, diploid yeast cells may divide by meiosis to form haploid cells, each with a different genetic constitution. This process is analogous to the formation of egg and sperm cells in higher eukaryotes. However, in yeast, the haploid cells appear identical, and there is no way to tell the sexes apart so we refer to mating types. In contrast to the haploid gametes of animals and plants, the haploid cells of yeast may grow and divide indefinitely in culture. Two haploid cells, of opposite mating types, may fuse to form a zygote.

Figure 1.19 Yeast Life Cycle

The yeast cell alternates between haploid and diploid phases and is capable of growth and cell division in either phase.

In its haploid phase, Saccharomyces cerevisiae has 16 chromosomes and nearly three times as much DNA as E. coli. Despite this, it only has 1.5 times as many genes as E. coli. It is easier to use the haploid phase of yeast for isolating mutations and analyzing their effects. Nonetheless, the diploid phase is also useful for studying how two alleles of the same gene interact in the same cell. Thus, yeast can be used as a model to study the diploid state and yet take advantage of its haploid phase for most of the genetic analysis.

Biotechnology is a new word but not a new occupation. Brewing and baking both use genetically-modified yeast that have been modified and selected for superior taste throughout history.

Yeast illustrates the genetic characteristics of higher organisms in a simplified manner.

Focus on Relevant Research

The list of model organisms presented in this chapter is only some of the actual model organisms used in laboratories across the world. Another eukaryotic model organism used in genetic research is Neurospora, a group of filamentous fungi. The associated article describes the attributes of Neurospora that make it an excellent model organism. First and foremost, Neurospora grows easily and quickly in the lab. The fungus has a haploid state and a well-defined sexual cycle. The genome is sequenced and thousands of different mutations have been isolated or created by mutagenesis. The most striking characteristic that makes Neurospora an interesting model organism is the ability to see the actual cells after meiosis. The ascospores stay in the same order in which they divided, so it is easy to follow how genes segregate during meiosis.

Selker, E (2011) Neurospora Curr. Biol. 21(4): R139–140.

8.4 A Roundworm and a Fly Are Model Multicellular Animals

If all the matter in the universe except the nematodes were swept away, our world would still be dimly recognizable…

—N.A. Cobb, 1914

Ultimately, researchers have to study multicellular creatures. The most primitive of these that is widely used is the roundworm, Caenorhabditis elegans (Fig. 1.20). Nematodes, or roundworms, are best known as parasites both of animals and plants. Although it is related to the eelworms—nematodes that attack the roots of crop plants—C. elegans is a free-living and harmless soil inhabitant that lives by eating bacteria. A single acre of soil in arable land may contain as many as 3,000 million nematodes belonging to dozens of different species.

Figure 1.20 C. elegans

The soil-dwelling nematode C. elegans shown with low-magnification phase contrast microscopy. C. elegans is convenient for genetic analysis because it is a hermaphrodite; that is, it makes sperm and eggs. It takes only three days to reach maturity and thousands of worms can be kept on a culture plate.(Credit: Jill Bettinger, Virginia Commonwealth University, Richmond, VA.)

The haploid genome of C. elegans consists of 97 Mb of DNA carried on six chromosomes. This is about seven times as much total DNA as in a typical yeast genome. C. elegans has an estimated 20,000 genes and so contains a much greater proportion of non-coding DNA than lower eukaryotes such as yeast. Its genes contain an average of four intervening sequences each.

The adult C. elegans is about 1 mm long and has 959 cells, and the lineage of each has been completely traced from the fertilized egg (i.e., the zygote). It is thus a useful model for the study of animal development. In particular, apoptosis, or programmed cell death, was first discovered and has since been analyzed genetically using C. elegans. Although very convenient in the special case of C. elegans, such a fixed number of cells in an adult multicellular animal is extremely rare. C. elegans, which lives about 2–3 weeks, is also used to study life span and the aging process. RNA interference, a gene-silencing technique that relies on double-stranded RNA, was discovered in C. elegans in 1998 and is now used to study gene function during development in worms and other higher animals. RNA interference is discussed in Chapter 18.

The fruit fly, Drosophila melanogaster (usually called Drosophila), was chosen for genetic analysis in the early part of the twentieth century (Fig. 1.21). Fruit flies live on rotten fruit and have a 2-week life cycle, during which the female lays several hundred eggs. The adults are about 3 mm long and the eggs about 0.5 mm. Once molecular biology came into vogue it became worthwhile to investigate Drosophila at the molecular level in order to take advantage of the wealth of genetic information already available. The haploid genome has 180 Mb of DNA carried on four chromosomes. Although we normally think of Drosophila as more advanced than a primitive roundworm, it has an estimated 14,000 genes—6,000 fewer than the roundworm, C. elegans. Research on Drosophila has concentrated on cell differentiation, development, signal transduction, and behavior.

Figure 1.21 Drosophila melanogaster , the Fruit Fly

Photo of a male Drosophila adult fly.(Credit: André Karwath, Creative Commons Attribution-Share Alike 2.5 Generic license, Wikipedia)

Nematodes that live in oceanic mud or inland soils may all look the same; nonetheless, they harbor colossal genetic diversity.

8.5 Zebrafish and Xenopus are used to Study Vertebrate Development

Danio rerio (previously Brachydanio rerio), the zebrafish, is increasingly being used as a model for studying genetic effects in vertebrate development. Zebrafish are native to the slow freshwater streams and rice paddies of East India and Burma, including the Ganges River. They are small, hardy fish, about an inch long that have been bred for many years by fish hobbyists in home aquariums where they may survive for about five years. The standard wild-type is clear-colored with black stripes that run lengthwise down its body (Fig. 1.22). Its eggs are laid in clutches of about 200. They are clear and develop outside the mother’s body, so it is possible to watch a zebrafish egg grow into a newly-formed fish under a microscope. Development from egg to adult takes about three months. Zebrafish are unusual in being nearly transparent so it is possible to observe the development of the internal organs.

Figure 1.22 Danio rerio

Danio rerio, the zebrafish, has recently been adopted as a model for the genetic study of embryonic development in higher animals.(Credit: James King-Holmes, SPL, Photo Researchers, Inc.)

Zebrafish have about 1,700 Mb of DNA on 25 chromosomes and most zebrafish genes have been found to be similar to human genes. The genome is now sequenced completely, which offers scientists another key element to study the organism. Genetic tagging is relatively easy and microinjecting the egg with DNA is straightforward. Consequently, the zebrafish has become a favorite model organism for studying the molecular genetics of embryonic development. In addition, zebrafish are able to regenerate their heart, nervous tissues, retina, hearing tissues, and fins, which can offer a glimpse into genes that control the growth of these tissues in humans also.

The use for zebrafish in molecular biology is growing. Zebrafish are now used in initial screening for new drugs. The fish and embryos absorb small molecules from the water, so to screen a drug for toxicity is fairly easy. Some screens use robotic microscopes to visualize embryos as they are exposed to new drugs. The microscopes can monitor thousands of embryos, exposed to thousands of new compounds. Compounds giving positive results are then used in mouse models and finally humans. Zebrafish are also able to grow human tumors. The tumor cells can be transplanted into a fish and each step of the tumor formation can be visualized because of the transparent nature of zebrafish.

Xenopus laevis, or the African clawed frog, is another key model organism for the understanding of vertebrate development (Fig. 1.23). These frogs live in any kind of water and are very easy to grow in the laboratory. Like zebrafish, Xenopus tadpoles develop outside the mother and are easily visualized throughout the entire developmental process. The size of the eggs allows researchers to inject different genes or chemical substances directly into the eggs. As the egg develops into a tadpole, the effect of this alteration can be visually determined.

Figure 1.23 Xenopus laevis , African Clawed Frog

dult Xenopus laevis is shown(Credit: Michael Linnenback; Wikipedia Commons.)

Box 1.06 Brainbow Fish

Late in 2003, zebrafish became the first commercially-available, genetically-engineered pets. Fluorescent red zebrafish are marketed in the United States by Yorktown Technologies as GloFish™. They fluoresce red when illuminated with white light, or better, black light (i.e., near UV) due to the presence of a gene for a red fluorescent protein taken from a sea coral. The principle is similar to that of the widely-used green fluorescent protein taken from jellyfish (see Ch. 19 for use of GFP in genetic analysis). The price of about $5 per fish makes GloFish™ about five times as expensive as normal zebrafish. The fish were developed at the National University of Singapore by researcher Zhiyuan Gong with the ultimate objective of monitoring pollution. A second generation of more specialized red fluorescent zebrafish will fluoresce in response to toxins or pollutants in the environment.

The ability to color zebrafish has now advanced. Recent work by Lichtman and Smith (2008) allows each individual cell to be labeled a different fluorescent color. The cells of the brain have been treated with over 90 different fluorescent colors, and each of the neurons is a different color, creating what scientists have called a brainbow (Fig. 1.24). The labeling provides researchers with the ability to visualize each and every neuron from the cell body all the way to its terminal branches. The different colors are crucial to follow the long axons of the neurons through their twists and turns in development. Using the color technology will enable scientists to trace each cell of the zebrafish through development, and create a lineage map similar to what has already been established in C. elegans.

Figure 1.24 Brainbow image of 5-day-old zebrafish embryo

This beautiful image of the brain of a 5-day-old zebrafish larva, which was created by Albert Pan of Harvard University, won 4th place in the 2008 Olympus BioScapes Digital Imaging competition.

8.6 Mouse and Man

The ultimate aim of molecular medicine is to understand human physiology at the molecular level and to apply this knowledge in curing disease. The complete sequence of the human genome is now known, but researchers have little idea of what the products of most of these genes actually do. Since direct experimentation with humans is greatly restricted, animal models are necessary. Although a range of animals has been used to investigate various topics, the rat and the mouse are the most widespread laboratory animals. Rats were favored in the early days of biochemistry when metabolic reactions were being characterized. Mice are smaller and breed faster than rats, and are easier to modify genetically. Consequently, the mouse is used more often for experiments involving genetics and molecular biology. Mice live from 1 to 3 years and become sexually mature after about 4 weeks. Pregnancy lasts about three weeks and may result in up to 10 offspring per birth.

Humans have two copies each of approximately 20,000–25,000 genes scattered over 23 pairs of chromosomes. Mice have a similar genome, of 2,600 Mb of DNA carried on 20 pairs of chromosomes. Less than 1% of mouse genes lack a homolog in the human genome. The average mouse (or human) gene extends over 40 kilobases of DNA that consists mostly of non-coding introns (approximately seven per gene). Nowadays there are many strains of mutant mice in which one or more particular genes have been altered or disrupted. These are used to investigate gene function (Fig. 1.25).

Figure 1.25 Transgenic Mice

The larger mouse contains an artificially introduced human gene, which causes a difference in growth. Mice with the human growth hormone gene grow larger than normal mice.(Credit Palmiter, et al. Nature 300: 611–615.)

Intact humans cannot be used for routine experiments for ethical reasons. However, it is possible to grow cells from both humans and other mammals in culture. Many cell lines from humans and monkeys are now available. Such cells are much more difficult to culture